The underlying hypothesis of this proposal is that most vertebrate organs begin their initial formation by a common, developmentally conserved pattern of inductive interactions between two tissues. The developing tooth germ is a prototype for such inductive tissue interactions and provides a powerful experimental system for elucidating the genetic pathways involved in organogenesis. Members of the Msx homeobox gene family are expressed at sites of epithelial-mesenchymal interactions during embryogenesis, including the tooth. We have prepared Msx1 and Msx2 knockout mice. Msx1 deficient mice exhibit an arrest in molar tooth development at the bud stage, while Msx2 deficient mice exhibit other epithelial-mesenchymal defects and only late defects in tooth development. Msx1-Msx2 double mutants, however, exhibit an arrest at the dental lamina stage of tooth development, indicating a requirement for Msx gene function in tooth formation at a distinct and earlier developmental step than that revealed by the Msx1 mutant. The co- expression of Msx, Bmp and Lef1 genes and the coincidence of tooth and other phenotypes between Msx and Lef1 knockout mice suggests that these genes reside within a genetic pathway. Preliminary results, obtained by analyzing Bmp4 and Lef1 expression in the Msx1 mutant molar tooth and by performing BMP4 bead implant experiments in wild-type and Msx1 mutant molar mesenchymes, indicate that Msx1 is required for the transmission of Bmp4 expression from dental epithelium to mesenchyme and also, indirectly, for Lef1 expression in dental mesenchyme. This grant proposes to further extend these findings in three ways. First, it is proposed to continue to identify and order additional genes responsible for early tooth development in a genetic pathway, using both the Msx1 and Msx1Msx2 double mutants. Second, we will test whether the Msx gene product can activate the expression of Bmp2 or Bmp4. Third, we will attempt to demonstrate that the regulation of Bmp genes by Msx homeoproteins is direct, by identifying potential high affinity Msx binding sites in these Bmp genes and by confirming the functional relevance of these sites in vivo by experiments involving transgenic mice. These studies will continue to build upon the genetic pathway already identified and permit the integration of additional gene products into the pathway. The results should provide fundamental insights into early tooth development which are also pertinent to other organs which form via epithelial-mesenchymal interactions.